The present invention relates to quinazoline derivatives, processes for their reparation, pharmaceutical compositions containing them as active ingredient, methods for the treatment of disease states associated with angiogenesis and/or increased vascular permeability, to their use as medicaments and to their use in the manufacture of medicaments for use in the production of antiangiogenic and/or vascular permeability reducing effects in warm-blooded animals such as humans.
Normal angiogenesis plays an important role in a variety of processes including embryonic development, wound healing and several components of female reproductive function. Undesirable or pathological angiogenesis has been associated with disease states including diabetic retinopathy, psoriasis, cancer, rheumatoid arthritis, atheroma, Kaposi""s sarcoma and haemangioma (Fan et al, 1995, Trends Pharmacol. Sci. 16: 57-66; Folknan, 1995, Nature Medicine 1: 27-31). Alteration of vascular permeability is thought to play a role in both normal and pathological physiological processes (Cullinan-Bove et al, 1993, Endocrinology 133: 829-837; Senger et al, 1993, Cancer and Metastasis Reviews, 12: 303-324). Several polypeptides with in vitro endothelial cell growth promoting activity have been identified including, acidic and basic fibroblast growth factors (aFGF and bFGF) and vascular endothelial growth factor (VEGF). By virtue of the restricted expression of its receptors, the growth factor activity of VEGF, in contrast to that of the FGFs, is relatively specific towards endothelial cells. Recent evidence indicates that VEGF is an important stimulator of both normal and pathological angiogenesis (Jakeman et al, 1993, Endocrinology, 133: 848-859; Kolch et al, 1995, Breast Cancer Research and Treatment, 36:139-155) and vascular permeability (Connolly et al, 1989, J. Biol. Chem. 264: 20017-20024). Antagonism of VEGF action by sequestration of VEGF with antibody can result in inhibition of tumour growth (Kim et al, 1993, Nature 362: 841-844).
Receptor tyrosine kinases (RTKs) are important in the transmission of biochemical signals across the plasma membrane of cells. These transmembrane molecules characteristically consist of an extracellular ligand-binding domain connected through a segment in the plasma membrane to an intracellular tyrosine kinase domain. Binding of ligand to the receptor results in stimulation of the receptor-associated tyrosine kinase activity which leads to phosphorylation of tyrosine residues on both the receptor and other intracellular molecules. These changes in tyrosine phosphorylation initiate a signalling cascade leading to a variety of cellular responses. To date, at least nineteen distinct RTK subfamilies, defined by amino acid sequence homology, have been identified. One of these subfamilies is presently comprised by the fms-like tyrosine kinase receptor, Flt or Flt1, the kinase insert domain-containing receptor, KDR (also referred to as Flk-1), and another fms-like tyrosine kinase receptor, Flt4. Two of these related RTKs, Flt and KDR, have been shown to bind VEGF with high affinity (De Vries et al, 1992, Science 255: 989-991; Terman et al, 1992, Biochem. Biophys. Res. Comm. 1992, 187: 1579-1586). Binding of VEGF to these receptors expressed in heterologous cells has been associated with changes in the tyrosine phosphorylation status of cellular proteins and calcium fluxes.
European Patent Publication No. 0326330 discloses certain quinoline, quinazoline and cinnoline plant fungicides. Certain of these plant fungicides are also stated to possess insecticidal and miticidal activity. There is however no disclosure or any suggestion that any of the compounds disclosed may be used for any purpose in animals such as humans. In particular, the European Patent Publication contains no teaching whatsoever concerning angiogenesis and/or increased vascular permeability mediated by growth factors such as VEGF.
European Patent Publication No. 0566226 discloses anilinoquinazolines which have activity against epidermal growth factor (EGF) receptor tyrosine kinase. EP 0566226 contains no teaching whatsoever concerning angiogenesis and/or increased vascular permeability mediated by growth factors such as VEGF. Moreover compounds of EP 0566226 which have been tested do not show significant activity against VEGF receptor tyrosine kinase.
The present invention is based on the surprising discovery that certain quinazolines inhibit the effects of VEGF, a property of value in the treatment of disease states associated with angiogenesis and/or increased vascular permeability such as cancer, diabetes, psoriasis, rheumatoid arthritis, Kaposi""s sarcoma, haemangioma, acute and chronic nephropathies, atheroma, arterial restenosis, autoimmune diseases, acute inflammation and ocular diseases with retinal vessel proliferation. Compounds of the present invention possess good activity against VEGF receptor tyrosine kinase whilst possessing some activity against EGF receptor tyrosine kinase. Furthermore, some compounds of the present invention, possess substantially higher potency against VEGF receptor tyrosine kinase than against EGF receptor tyrosine kinase or FGF R1 receptor tyrosine kinase. Thus certain compounds of the invention which have been tested possess activity against VEGF receptor tyrosine kinase such that they may be used in an amount sufficient to inhibit VEGF receptor tyrosine kinase whilst demonstrating no significant activity against EGF receptor tyrosine kinase or FGF R1 receptor tyrosine kinase. While we do not wish to be bound by theoretical considerations such compounds may for example be of interest in treating tumours which are associated with VEGF, especially those tumours which are dependent on VEGF for their growth.
Other compounds of the invention possess good activity against both VEGF and EGF receptor tyrosine kinases. Indeed certain compounds possess substantially equivalent activities against VEGF and EGF receptor tyrosine kinases. It is believed that these compounds may be of interest in treating tumour states associated with both VEGF and EGF, especially where a patient is suffering from a condition in which tumours are present which are dependent on both VEGF and EGF for their growth.
According to one aspect of the present invention there is provided a quinazoline derivative of the formula I: 
wherein:
m is an integer from 1 to 2;
R1 represents hydrogen, hydroxy, halogeno, nitro, trifluoromethyl, cyano, C1-3alkyl, C1-3alkoxy, C1-3alkylthio, or xe2x80x94NR5R6 (wherein R5 and R6, which may be the same or different, each represents hydrogen or C1-3alkyl);
R2 represents hydrogen, hydroxy, halogeno, methoxy, amino or nitro:
R3 represents hydroxy, halogeno, C1-3alkyl, C1-3alkoxy, C1-3alkanoyloxy, trifluoromethyl, cyano, amino or nitro;
X1 represents xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR7COxe2x80x94, xe2x80x94CONR8xe2x80x94, xe2x80x94SO2NR9xe2x80x94, xe2x80x94NR10SO2xe2x80x94 or xe2x80x94NR11xe2x88x92 (wherein R7, R8, R9, R10 and R11 each independently represents hydrogen, C1-3alkyl or C1-3alkoxyC2-3alkyl);
R4 is selected from one of the following eight groups:
1) C1-5alkylR12 (wherein R12 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group is linked to C1-5alkyl through a carbon atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl) or C1-5alkylR13 (wherein R13 is a group selected from pyrrolidin-1-yl, imidazolidin-1-yl and thiomorpholino, which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
2) C2-5alkenylR14 (wherein R14 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
3) C2-5alkynylR15 (wherein R15 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
4) C1-5alkylX2C1-5alkylX3R16 (wherein X2 and X3 which may be the same or different are eachxe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR17COxe2x80x94, xe2x80x94CONR18xe2x80x94, xe2x80x94SO2NR19xe2x80x94, xe2x80x94NR20SO2xe2x80x94 or xe2x80x94NR21xe2x80x94 (wherein R17, R18, R19, R20 and R21 each independently represents hydrogen, C1-3alkyl or C1-3alkoxyC2-3alkyl) and R16 represents hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C1-5alkylX2C1-5alkylX3R16;
5) C1-5alkylX4COR22 (wherein X4 represents xe2x80x94Oxe2x80x94 or xe2x80x94NR23xe2x80x94 (wherein R23 represents hydrogen, C1-3alkyl or C1-3alkoxyC2-3alkyl) and R22 represents xe2x80x94NR24R25 or xe2x80x94OR26 (wherein R24, R25 and R26 which may be the same or different each represents hydrogen, C1-4alkyl or C1-3alkoxyC2-3alkyl));
6) C1-5alkylX5R27 (wherein X5 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2, xe2x80x94OCOxe2x80x94, xe2x80x94NR28COxe2x80x94, xe2x80x94CONR29xe2x80x94, xe2x80x94SO2NR30xe2x80x94, xe2x80x94NR31SO2xe2x80x94 or xe2x80x94NR32xe2x80x94 (wherein R28, R29, R30, R31 and R32 each independently represents hydrogen, C1-3alkyl or C1-3alkoxyC2-3alkyl) or X5 is carbonyl, and R27 represents cyclopentyl, cyclohexyl or a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which cyclopentyl, cyclohexyl or heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl or R27 is C1-3alkyl with the proviso that when R27 is C1-3alkyl, X5 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR30xe2x80x94or xe2x80x94NR31SO2xe2x80x94 and X1 is not xe2x80x94CH2xe2x80x94);
7) C1-3alkoxyC2-4alkyl provided that X1 is xe2x80x94Sxe2x80x94, or X1 is xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94; and
8) C1-3alkoxyC2-4alkyl or C1-4alkyl provided that X1 is xe2x80x94Oxe2x80x94; and additionally R4 may be selected from the following five groups:
9) C1-5alkylX6C1-5alkylR33 (wherein X6 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR34COxe2x80x94, xe2x80x94CONR35xe2x80x94SO2NR36xe2x80x94, xe2x80x94NR37SO2xe2x80x94 or xe2x80x94NR38xe2x80x94 (wherein R34, R35, R36 R37 and R38 each independently represents hydrogen, C1-3alkyl or C1-3alkoxyC2-3alkyl) and R33 represents cyclopentyl, cyclohexyl or a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which cyclopentyl, cyclohexyl or heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl, C1-4alkoxy, carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
10) R39 (wherein R39 is a group selected from pyrrolidin-3-yl, piperidin-3-yl and piperidin4-yl which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl, C1-4alkoxy, carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
11) C1-5alkylR40 (wherein R40 is piperazin-1-yl which bears at least one substituent selected from C1-4alkanoyl, C1-4alkoxycarbonyl, C1-4hydroxyalkyl and xe2x80x94CONR41R42 (wherein R41 and R42 each independently represents hydrogen or C1-4alkyl);
12) C1-5alkylR43 (wherein R43 is morpholino which may bear one or two substituents selected from oxo, C1-4alkyl, C1-4hydroxyalkyl, carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl) with the proviso that when R4 is C1-5alkylR43, X1 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR9xe2x80x94 or xe2x80x94NR10SO2xe2x80x94; and
13) C1-5alkylR44 (wherein R44 is morpholino which bears at least one and optionally two substituents selected from oxo, C1-4alkyl, C1-4hydroxyalkyl, carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
with the further proviso that when R4 is selected from group 8) R1 and/or R2 is/are nitro or at least one R3 is C1-3alkanoyloxy;]
and salts thereof.
Preferably m is 2.
R1 is advantageously hydrogen, hydroxy, cyano, nitro, trifluoromethyl, C1-3alkyl, C1-3alkoxy or amino.
R1 is preferably hydrogen, hydroxy, cyano, nitro, trifluoromethyl, methyl, ethyl, methoxy or ethoxy, more preferably hydrogen, methyl or methoxy, most preferably hydrogen or methoxy, but especially methoxy.
R2 is preferably hydrogen, fluoro, amino or nitro, but especially hydrogen.
In one embodiment of the present invention R3 represents hydroxy, halogeno, C1-2alkyl, C1-2alkoxy, trifluoromethyl, cyano, amino or nitro.
Advantageously in another embodiment of the present invention one R3 substituent is metahydroxy and the other one is selected from halogeno and methyl.
In another embodiment of the invention the phenyl group bearing (R3)m is preferably of the formula II: 
wherein:
Ra represents hydrogen, methyl, fluoro or chloro, preferably hydrogen or fluoro;
Rb represents hydrogen, methyl, methoxy, bromo, fluoro or chloro, especially hydrogen, methyl or chloro;
RC represents hydrogen or hydroxy;
Rd represents hydrogen, fluoro or chloro, especially hydrogen or fluoro.
In a particular aspect of the present invention, the phenyl group bearing (R3), is the 3-hydroxy4-methylphenyl group, the 2-fluoro-5-hydroxyphenyl group or the 4-chloro-2-fluorophenyl group, or the 4-bromo-2-fluorophenyl group, especially the 4-chloro-2-fluorophenyl group or the 4-bromo-2-fluorophenyl group more especially the 4-chloro-2-fluorophenyl group.
Advantageously X1 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NR7COxe2x80x94, xe2x80x94NR10S2xe2x80x94 or xe2x80x94NR11xe2x80x94 (wherein R7, R10 and R11 each independently represents hydrogen, C1-2alkyl or C1-2alkoxyethyl).
Preferably X1 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NR7COxe2x80x94or xe2x80x94NR10SO2xe2x80x94 (wherein R7 and R10 each independently represents hydrogen or C1-2alkyl).
More preferably X1 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NR7COxe2x80x94 (wherein R7 represents hydrogen or methyl).
Particularly X1 represents xe2x80x94Oxe2x80x94, or xe2x80x94NHCOxe2x80x94, or xe2x80x94Sxe2x80x94, especially xe2x80x94Oxe2x80x94, or xe2x80x94Sxe2x80x94, more especially xe2x80x94Oxe2x80x94.
Conveniently X2 and X3 which may be the same or different each represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR17COxe2x80x94, or xe2x80x94NR21xe2x80x94 (wherein R17 and R21 each independently represents hydrogen, C1-2alkyl or C1-2alkoxyethyl).
Advantageously X2 and X3 which may be the same or different each represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94 or xe2x80x94NR21xe2x80x94 (wherein R22 represents hydrogen, C1-2alkyl or C 1-2alkoxyethyl).
Preferably X2 and X3 which may be the same or different each represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94NR21xe2x80x94 (wherein R21 represents hydrogen, C1-2alkyl or C1-2alkoxyethyl).
In a particular aspect of the present invention X3 is xe2x80x94Oxe2x80x94 and X2 is xe2x80x94NR17COxe2x80x94 (wherein R17 represents hydrogen, or methyl).
Advantageously X4 represents xe2x80x94Oxe2x80x94 or xe2x80x94NR23xe2x80x94 (wherein R23 represents hydrogen, C1-3alkyl or C1-2alkoxyethyl).
Advantageously X5 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR28COxe2x80x94, xe2x80x94NR31SO2xe2x80x94 or xe2x80x94NR32xe2x80x94 (wherein R28, R31 and R32 each independently represents hydrogen, C1-2alkyl or C1-2alkoxyethyl) or X5 is carbonyl.
Preferably X5 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94 or xe2x80x94NR32xe2x80x94 (wherein R32 represents hydrogen, C1-2alkyl or C1-2alkoxyethyl).
More preferably X5 represents xe2x80x94Oxe2x80x94 or xe2x80x94NR32xe2x80x94 (wherein R32 represents hydrogen or C1-2alkyl).
Advantageously X6 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR34COxe2x80x94, xe2x80x94NR34SO2xe2x80x94 or xe2x80x94NR38xe2x80x94 (wherein R34, R37 and R38 each independently represents hydrogen, C1-2alkyl or C1-2alkoxyethyl).
Preferably X6 represents xe2x80x94Oxe2x80x94.
Conveniently R4 is selected from one of the following eight groups:
1) C1-5alkylR12 (wherein R12 is a group selected from 1,3-dioxolan-2-yl, 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, 1,3-dithian-2-yl, pyrrolidin-2-yl and pyrrolidin-3-yl, and piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl and piperazin-2-yl which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl) or C2-5alkylR45 (wherein R45 is a group selected from imidazolidin-1-yl, pyrrolidin-1-yl and thiomorpholino, which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
2) C3-5alkenylR46 (wherein R46 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group is linked to C3-5alkenyl through a carbon atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl) or C4-5alkenylR47 (wherein R47 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms of which one is N and the other is selected independently from O, S and N, which heterocyclic group is linked to C4-5alkenyl through a nitrogen atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
3) C3-5alkynylR48 (wherein R48 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group is linked to C3-5alkynyl through a carbon atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl) or C4-5alkynylR49 (wherein R49 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms of which one is N and the other is selected independently from O, S and N, which heterocyclic group is linked to C4-5alkynyl through a nitrogen atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl and C1-4alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
4) C2-3alkylX2C2-3alkylX3R16 (wherein X2 and X3 are as defined hereinbefore and R16 represents hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2C2-3alkylX3R16;
5) C2-3alkylX4COR22 (wherein X4 is as defined hereinbefore and R22 represents xe2x80x94NR24R25 or xe2x80x94OR26 (wherein R24, R25 and R26 which may be the same or different each represents hydrogen, C1-4alkyl or C1-2alkoxyethyl));
6) C2-4alkylX5R27 (wherein X5 is as defined hereinbefore and R27 represents cyclopentyl, cyclohexyl or a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which cyclopentyl, cyclohexyl or heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C, alkoxycarbonyl or R27 is C1-3alkyl with the proviso that when R27 is C1-3alkyl, X5 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR30xe2x80x94 or xe2x80x94NR31SO2xe2x80x94 and X1 is not xe2x80x94CH2xe2x80x94);
7) C1-3alkoxyC2-4alkyl provided that X1 is xe2x80x94Sxe2x80x94, or X1 is xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94; and
8) C1-3alkoxyC2-4alkyl or C1-4alkyl provided that X1 is xe2x80x94Oxe2x80x94; and additionally R4 may conveniently be selected from the following four groups:
9) C2-4alkylX6C2-4alkylR33 (wherein X6 is as defined hereinbefore and R33 represents a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-4alkyl, C1-4hydroxyalkyl, C1-4alkoxy, carbamoyl, C1-4alkylcarbamoyl, N,N-di(C1-4alkyl)carbamoyl, C1-4alkanoyl and C1-4alkoxycarbonyl);
10) C2-4alkylR40 (wherein R40 is piperazin-1-yl which bears at least one substituent selected from C2-3alkanoyl, C1-3alkoxycarbonyl, C1-3hydroxyalkyl and CONR41R42 (wherein R41 and R42 each independently represents hydrogen or C1-3alkyl));
11) C2-4alkylR43 (wherein R43 is morpholino which may bear one or two substituents selected from oxo. C1-3alkyl, C1-3hydroxyalkyl, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl) with the proviso that when R4 is C2-4alkylR43, X1 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR9xe2x80x94 or xe2x80x94NR10SO2xe2x80x94; and
12) C2-4alkylR44 (wherein R44 is morpholino which bears at least one and optionally two substituents selected from oxo, C1-3alkyl, C1-3hydroxyalkyl, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl);
with the further proviso that when R4 is selected from group 8) R1 and/or R2 is/are nitro or at least one R3 is C1-3alkanoyloxy.
An additional convenient value of R4 is 2-3alkylX2methylX3R16 (wherein X2 and X3 are as defined hereinbefore and R16 represents hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2methylX3R16.
Advantageously R4 is selected from one of the following seven groups:
1) C1-4alkylR12 (wherein R12 is a group selected from 1,3-dioxolan-2-yl, 1,3-dioxan-2-yl, 1.3-dithiolan-2-yl, 1,3-dithian-2-yl, pyrrolidin-2-yl and pyrrolidin-3-yl, and piperidin-2-yl, piperidin-3-yl, piperidin4-yl, morpholin-2-yl, morpholin-3-yl and piperazin-2-yl which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl) or C2-4alkylR45 (wherein R45 is a group selected from imidazolidin-1-yl, pyrrolidin-1-yl and thiomorpholino which group may bear one or two substituents selected from oxo, hydroxy halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C1-3alkanoyl and C1-3alkoxycarbonyl);
2) 1-R46prop-1-en-3-yl, 1-R46but-2-en-4-yl, 1-R46but-1-en-3-yl, 1-R46pent-2-en-4-yl or 2-R46pent-3-en-5-yl (wherein R46 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group is linked to the alkenyl group through a carbon atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl) or 1-R47but-2-en-4-yl, 1-R47pent-2-en-4-yl or 2-R47 pent-3-en-5-yl (wherein R47 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, of which one is N and the other is selected independently from O, S and N, which heterocyclic group is linked to the alkenyl group through a nitrogen atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl);
3) 1-R48prop-1-yn-3-yl, 1-R48but-2-yn-4-yl, 1-R48but-1-yn-3-yl, 1-R48pent-2-yn-4 or 2-R48pent-3-yn-5-yl (wherein R48 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group is linked to the alkynyl group through a carbon atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl) or 1-R49but-2-yn-4-yl, 1-R49pent-2-yn-4-yl or 2-R49pent-3-yn-5-yl (wherein R49 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, of which one is N and the other is selected independently from O, S and N, which heterocyclic group is linked to the alkynyl group through a nitrogen atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl);
4) C2-3alkylX2C2-3alkylX3R16 (wherein X2 and X3 are as defined hereinbefore and R16 represents hydrogen or C1-3allyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2C2-3alkylX3R16;
5) C2-3alkylX4COR22 (wherein X4 is as defined hereinbefore and R22 represents xe2x80x94NR24R25 or xe2x80x94OR26 (wherein R24, R25 and R26 which may be the same or different each represents hydrogen, C1-4alkyl or C1-2alkoxyethyl));
6) C2-3alkylX5R27 (wherein X5 is as defined hereinbefore and R27 represents a group selected from cyclopentyl, cyclohexyl, pyrrolidinyl and piperidinyl which group is linked to X5 through a carbon atom and which group may carry one substituent selected from oxo, hydroxy, halogeno, C1-2alkyl, C1-2hydroxyalkyl and C1-2alkoxy, and additional possible substituents are carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl or R27 is C1-3alkyl with the proviso that when R27 is C1-3alkyl, X5 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR30xe2x80x94 or xe2x80x94NR31SO2xe2x80x94 and X1 is not xe2x80x94CH2xe2x80x94); and
7) C1-2alkoxyC2-3alkyl provided that X1 is xe2x80x94Sxe2x80x94, or X1 is xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94; and additionally R4 may advantageously be selected from the following four groups:
8) C2-3alkylX6C2-3alkylR33 (wherein X6 is as defined hereinbefore and R33 represents a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O. S and N, which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl, C1-3alkoxy, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl, and C1-3alkoxycarbonyl);
9) C2-3alkylR40 (wherein R40 is piperazin-1-yl which bears at least one substituent selected from acetyl, C1-2alkoxycarbonyl, C1-2hydroxyalkyl and CONR41R42 (wherein R41 and R42 each independently represents hydrogen or C1-2alkyl);
10) C2-3alkylR43 (wherein R43 is morpholino which may bear one or two substituents selected from oxo, C1-2alkyl, C1-2hydroxyalkyl, carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl) with the proviso that when R4 is C2-3 alkylR43, X1 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR9xe2x80x94 or xe2x80x94NR10SO2xe2x80x94; and
11) C2-3alkylR44 (wherein R44 is morpholino which bears at least one and optionally two substituents selected from oxo, C1-2alkyl, C1-2hydroxyalkyl, carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl).
An additional advantageous value of R4 is C2-3alkylX2methylX3R16 (wherein X2 and X3 are as defined hereinbefore and R16 represents hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2methylX3R16.
Preferably R4 is selected from one of the following seven groups:
1) C1-3alkylR2 (wherein R12 is a group selected from 1,3-dioxolan-2-yl, 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, 1,3-dithian-2-yl, pyrrolidin-2-yl and pyrrolidin-3-yl and piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl and piperazin-2-yl which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, acetyl and C1-3alkoxycarbonyl) or C2-3alkylR45 (wherein R45 is a group selected from imidazolidin-1-yl, pyrrolidin-1-yl and thiomorpholino which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl. C1-3hydroxyalkyl and C1-3alkoxy and additional possible substituents are carbamoyl, C1-3 alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, acetyl and C1-3alkoxycarbonyl);
2)1-R46but-2-en-4-yl (wherein R46 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group is linked to the alkenyl group through a carbon atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, acetyl and C1-3alkoxycarbonyl) or 1-R47but-2-en-4yl (wherein R47 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, of which one is N and the other is selected independently from O, S and N, which heterocyclic group is linked to the alkenyl group through a nitrogen atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, acetyl and C1-3alkoxycarbonyl);
3) 1-R48 but-2-yn-4-yl (wherein R48 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group is linked to the alkynyl group through a carbon atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3,hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, acetyl and C1-3alkoxycarbonyl) or 1-R49but-2-yn4-yl (wherein R49 is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, of which one is N and the other is selected independently from O, S and N, which heterocyclic group is linked to the alkynyl group through a nitrogen atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl and C1-3alkoxy, and additional possible substituents are carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, acetyl and C1-3alkoxycarbonyl);
4) C2-3alkylX2C2-3alkylX3R16 (wherein x2 and X3 are as defined hereinbefore and R16 represents hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2C2-3alkylX3R16;
5) 2-(3,3-dimethylureido)ethyl, 3-(3,3-dimethylureido)propyl, 2-(3-methylureido)ethyl, 3-(3-methylureido)propyl, 2-ureidoethyl, 3-ureidopropyl, 2-(N,N-dimethylcarbamoyloxy)ethyl, 3-(N,N-dimethylcarbamoyloxy)propyl, 2-(N,-methylcarbamoyloxy)ethyl, 3-(N, methylcarbamoyloxy)propyl, 2-(carbamoyloxy)ethyl, 3-(carbamoyloxy)propyl or 2-(1,3,3-trimethylureido)ethyl, 3-(1,3,3-trimethylureido)propyl, 2-(isopropoxycarbonylamino)ethyl, 3-(isopropoxycarbonylamino)propyl, 2-(isobutoxycarbonylamino)ethyl, 3-(isobutoxycarbonylamino)propyl, 2-(t-butoxycarbonylamino)ethyl or 3-(t-butoxycarbonylamino)propyl;
6) C2-3alkylX5R27 (wherein X5 is as defined hereinbefore and R27 is a group selected from cyclopentyl, cyclohexyl, pyrrolidinyl and piperidinyl which group is linked to X5 through a carbon atom and which group may carry one substituent selected from oxo, hydroxy, halogeno, C1-2alkyl, C1-2hydroxyalkyl and C1-2alkoxy and additional possible substituents are carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl or R27 is C1-2alkyl with the proviso that when R27 is C1-2alkyl, X5 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR30xe2x80x94 or xe2x80x94NR31SO2xe2x80x94 and X1 is not xe2x80x94CH2xe2x80x94); and
7) C1-2alkoxyC2-3alkyl provided that X1 is xe2x80x94Sxe2x80x94, or X1 is xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94; and additionally R4 may preferably be selected from the following three groups:
8) C2-3alkylX6C2-3alkylR33 (wherein X6 is as defined hereinbefore and R33 represents a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O. S and N, of which at least one is N, which heterocyclic group is linked to C2-3alkyl through a nitrogen atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-2alkyl, C1-2hydroxyalkyl, C1-2alkoxy, carbamoyl, C1-2alkylcarbamoyl, N,Ndi(C1-2carbamoyl, acetyl and C1-2alkoxycarbonyl);
9) C2-3alkylR43 (wherein R13 is morpholino which may bear one or two substituents selected from oxo, C1-2alkyl, C1-2hydroxyalkyl, carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl) with the proviso that when R4 is C2-3alkylR43, X1 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR9xe2x80x94 or xe2x80x94NR10SO2xe2x80x94); and
10) C2-3alkylR44 (wherein R44 is morpholino which bears at least one and optionally two substituents selected from oxo, C1-2alkyl, C1-2hydroxyalkyl, carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl).
An additional preferred value of R4 is C2-3alkylX2methylX3R16 (wherein X2 and X3 are as defined hereinbefore and R16 represents hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2methylX3R16.
More preferably R4 is selected from one of the following five groups:
1) C1-3alkylR12 (wherein R12 is a group selected from 1,3-dioxolan-2-yl, 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, 1,3-dithian-2-yl, pyrrolidin-2-yl and pyrrolidin-3-yl and piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl morpholin-3-yl and piperazin-2-yl which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-2alkyl, C1-2hydroxyalkyl and C1-2alkoxy, and additional possible substituents are carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl) or C2-3alkylR45 (wherein R45 s is a group selected from imidazolidin-1-yl, pyrrolidin-1-yl and thiomorpholino which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-2alkyl, C1-2hydroxyalkyl and C1-2alkoxy, and additional possible substituents are carbamoyl. C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl);
2) 1-R50but-2-en-4-yl (wherein R50 is a group selected from imidazolidin-1-yl, 1,3-dioxolan-2-yl, 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, 1,3-dithian-2-yl, piperidin-4-yl, pyrrolidin-1-yl, pyrrolidin-3-yl, piperazin-1-yl, morpholino and thiomorpholino and piperidino which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-2alkyl, C1-2hydroxyalkyl and C1-2alkoxy, and additional possible substituents are carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl);
3)1-R51but-2-yn4-yl (wherein R51 is a group selected from imidazolidin-1-yl, 1,3dioxolan-2-yl, 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, 1,3-dithian-2-yl, piperidin4-yl, pyrrolidin-1-yl, pyrrolidin-3-yl, piperazin-1-yl, morpholino and thiomorpholino and piperidino which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-2alkyl, C1-2hydroxyalkyl and C1-2alkoxy, and additional possible substituents are carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl);
4) C2-3alkylX2C2-3alkylX3R16 (wherein X2 and X3 are as defined hereinbefore and R16 represent hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2C2-3alkylX3R16; and
5) C1-2alkoxyC2-3alkyl provided that X1 is xe2x80x94Sxe2x80x94, or X1 is xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94; and additionally R4 may more preferably be selected from the following four groups:
6) 2-(3,3-dimethylureido)ethyl, 3-(3,3-dimethylureido)propyl, 2-(3-methylureido)ethyl, 3-(3-methylureido)propyl, 2-ureidoethyl, 3-ureidopropyl, 2-(N,N-dimethylcarbamoyloxy)ethyl, 3-(N,N-dimethylcarbamoyloxy)propyl, 2-(N-methylcarbamoyloxy)ethyl, 3-(N-methylcarbamoyloxy)propyl, 2-(carbamoyloxy)ethyl, 3-(carbamoyloxy)propyl, 2-(1,3,3-trimethylureido)ethyl, 3-(1,3,3-trimethylureido)propyl, 2-(isopropoxycarbonylamino)ethyl. 3-(isopropoxycarbonylamino)propyl, 2-(isobutoxycarbonylamino)ethyl, 3-(isobutoxycarbonylamino)propyl, 2-(t-butoxycarbonylamino)ethyl or 3-(t-butoxycarbonylamino)propyl;
7) C2-3alkylX5R27 (wherein R27 is C1-2alkyl and X5 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR30xe2x80x94 or xe2x80x94NR31SO2xe2x80x94 and with the proviso that X1 is not xe2x80x94CH2xe2x80x94);
8) C2-3alkylX6C2-3alkylR33 (wherein X6 is as defined hereinbefore and R33 represent a group selected from morpholino, 2-oxopyrrolidin-1-yl, pyrrolidin-1-yl, piperidino, piperazin-1-yl and 4-methylpiperazin-1-yl); and
9) C2-3alkylR43 (wherein R43 is morpholino which may bear one or two substituents selected from oxo, C1-2alkyl, C1-2hydroxyalkyl, carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl) with the proviso that when R4 is C2-3alkylR43, X1 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR9xe2x80x94 or xe2x80x94NR10SO2xe2x80x94.
An additional more preferred value of R4 is C2-3alkylX2methylX3R16 (wherein X2 and X3 are as defined hereinbefore and R16 represents hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2methylX3R16.
Most preferably R4 is selected from one of the following five groups:
1) C1-3alkylR12 (wherein R12 is 1,3-dioxolan-2-yl, 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, 1,3-dithian-2-yl, pyrrolidin-2-yl or pyrrolidin-3-yl or piperidin-2-yl, piperidin-3-yl, piperidin4-yl, 1-methylpiperidin-2-yl, 1-methylpiperidin-3-yl, 1-methylpiperidin4-yl, 1-methylpyrrolidin-2 yl, 1-methylpyrrolidin-3-yl, piperazin-2-yl, 1-methylpiperazin-2-yl, 4-methylpiperazin-2-yl, 1,4-dimethylpiperazin-2-yl, morpholin-2-yl, morpholin-3-yl, 4-methylmorpholin-2-yl or 4-methylmorpholin-3-yl) or C2-3alkylR45 (wherein R45 is pyrrolidin-1-yl or thiomorpholino or 1,1-dioxothiomorpholino, 2-oxopyrrolidin-1-yl, 2-(N-methylcarbamoyl)pyrrolidin-1-yl, 2xe2x80x94(N,N-dimethylcarbamoyl)pyrrolidin-1-yl, 2-carbamoylpyrrolidin-1-yl, 2-oxoimidazolidin-1-yl or 3-methyl-2-oxoimidazolidin-1-yl);
2) 1-R50but-2-en-4-yl (wherein R50 is 2-oxoimidazolidin-1-yl, 1,3-dioxolan-2-yl, 1,3-2-yl, 1,3-dithiolan-2-yl, 1,3-dithian-2-yl, piperidin-4-yl, 1-methylpiperidin-4-yl, pyrrolidin-1-yl, 1-methylpyrrolidin-3-yl, piperazin-1-yl, morpholino or thiomorpholino or 4-methylpiperazin-1-yl, piperidino or 3-methyl-2-oxoimidazolidin-1-yl);
3) 1-R51but-2-yn-4-yl (wherein R51 is 2-oxoimidazolidin-1-yl, 1,3-dioxolan-2-yl, 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, 1,3-dithian-2-yl, piperidin4-yl, 1-methylpiperidin-4-yl, pyrrolidin-1-methylpiperidin-4-yl, pyrrolidin-1-yl, 1-methylpyrrolidin-3-yl, piperazin-1-yl, morpholino or thiomorpholino or 4-methylpiperazin-1-yl, piperidino or 3-methyl-2-oxoimidazolidin-1-yl);
4) C2-3alkyl X2C2-3alkylX3R16 (wherein X2 and X3 are as defined hereinbefore and R16 represents hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2C2-3alkylX3R16; and
5) C1-2alkoxyC2-3alkyl provided that X1 is xe2x80x94Sxe2x80x94, or X1 is xe2x80x94SOxe2x80x94or xe2x80x94SO2xe2x80x94; and additionally R4 may most preferably be selected from the following three groups:
6) C2-3alkylX5R27 (wherein R27 is C1-2alkyl and X5 is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR30xe2x80x94 or xe2x80x94NR31SO2xe2x80x94 and with the proviso that X1 is not xe2x80x94CH2xe2x80x94);
7) C2-3alkylX6C2-3alkylR33 (wherein X6 is as defined hereinbefore and R33 represent a group selected from pyrrolidin-1-yl, 4-methylpiperazin-1-yl and morpholino); and
8) C2-3alkylR43 (wherein R43 is morpholino which may bear one or two substituents selected from oxo, C1-2alkyl, C1-2hydroxyalkyl) with the proviso that when R4 is C2-3alkylR43, X43 is xe2x80x94S xe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NR9xe2x80x94 or xe2x80x94NR10SO2xe2x80x94.
An additional most preferred value of R4 is C1-3alkylX2methylX3R16 (wherein x2 and X3 are as defined hereinbefore and R16 represents hydrogen or C1-3alkyl) with the proviso that X1 cannot be xe2x80x94CH2xe2x80x94 when R4 is C2-3alkylX2methylX3R16.
Especially preferred values for the group R4-X1 are 3-(methylsulphonyl)propoxy, (1-methylpiperidin-3-yl)methoxy, 4-(pyrrolidin-1-yl)but-2-en-1-yloxy, 2-(2-methoxyethoxy)ethoxy, 3-(1,1-dioxothiomorpholino)propoxy, 2-(2-(pyrrolidin-1-yl)ethoxy)ethoxy, 2-(2-(4-methylpiperazin-1-yl)ethoxy)ethoxy, 3-morpholinopropylthio, 2-([N-methoxyacetyl-N-methyl]amino)ethoxy, 2-(2-oxopyrrolidin-1-yl)ethoxy, 2-thiomorpholinoethoxy, 3-(2-carbamoylpyrrolidin-1-yl)propoxy, 3-(2-oxopyrrolidin-1-yl)propoxy and 2-(2-morpholinoethoxy)ethoxy.
More especially preferred values for the group R4-X1 are 3-(methylsulphonyl)propoxy, (1-methylpiperidin-3-yl)methoxy and 4-(pyrrolidin-1-yl)but-2-en-1-yloxy.
In a particular aspect of the present invention there is provided a compound of the formula Ia: 
[wherein:
R1a is hydrogen or methoxy;
R2a is hydrogen;
the phenyl group bearing (R3a)ma is the 4-chloro-2-fluorophenyl group or the 4-bromo-2-fluorophenyl group;
X1a is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NR5aCOxe2x80x94 or NR6aSO2xe2x80x94 (wherein R5a and R6a each independently represents hydrogen or C1-2alkyl);
R4a is selected from one of the following eleven groups:
1) C1-4alkylR7a (wherein R7a is a group selected from 1,3-dioxolan-2-yl. 1,3-dioxan-2-yl, 1,3-dithiolan-2-yl, 1,3-dithian-2-yl, pyrrolidin-2-yl and pyrrolidin-3-yl, and piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl and piperazin-2-yl which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl, C1-3alkoxy, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl) or C2-4alkylR8a (wherein R8a is a group selected from imidazolidin-1-yl, pyrrolidin-1-yl and thiomorpholino which group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl, C1-3alkoxy, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl);
2) 1-R9aprop-1-en-3-yl, 1-R9abut-2-en4-yl, 1-R9abut-1-en-3-yl, 1-R9apent-2-en-4- or 2R9pent-3-en-5-yl (wherein R9ais a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group is linked to the alkenyl group through a carbon atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl, C1-3alkoxy, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, 2-3alkanoyl and C1-3alkoxycarbonyl) or 1-R10a but-2-en-4-yl, 1-R10apent-2-en-4-yl or 2-R10apent-3-en-5-yl(wherein R10a is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, of which one is N and the other is selected independently from O, S and N, which heterocyclic group is linked to the alkenyl group through a nitrogen atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl. C1-3alkoxy, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2 alkanoyl and C1-3alkoxycarbonyl);
3) 1-R11aprop-1-yn-3-yl, 1-R11abut-2-yn4-yl, 1-R11abut-1-yn-3-yl, 1-R11apent-2-yn-4yl or 2-R11apent-3-yn-5-yl (wherein R11a is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group is linked to the alkynyl group through a carbon atom and which heterocyclic group may bear one or two substituents selected from oxo; hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl, C1-3alkoxy, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C, alkyl)carbamoyl, C2,alkanoyl and C1-3alkoxycarbonyl) or 1-R12abut-2-yn4-yl, 1-R12apent-2-yn4-yl or 2-R12apent-3-yn-5 (wherein R12a is a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, of which one is N and the other is selected independently from O, S and N, which heterocyclic group is linked to the alkynyl group through a nitrogen atom and which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl, C1-3alkoxy, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl and C1-3alkoxycarbonyl);
4) C2-3alkylX2aC2-3alkylX3aR13a (wherein X2a and X3a which may be the same or different each represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, NR14aCOxe2x80x94, or xe2x80x94NR15axe2x80x94 (wherein R14a and R15a each independently represents hydrogen, C1-2alkyl or C1-2alkoxyethyl) and R13a represents hydrogen or C1-3alkyl);
5) C2-3alkylX4aCOR16 (wherein X4a represents xe2x80x94Oxe2x80x94 or xe2x80x94NR17axe2x80x94 (wherein R17a represents hydrogen, C1-3alkyl or C1-2alkoxyethyl) and R16a represents xe2x80x94NR18aR19a or xe2x80x94OR20a (wherein R18a, R19a and R20a which may be the same or different each represents hydrogen, C1-4alkyl or C1-2alkoxyethyl));
6) C1-3alkylX5aR5a (wherein X5a represents carbonyl, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR22aCOxe2x80x94, xe2x80x94NR23aSO2xe2x80x94 or xe2x80x94NR24axe2x80x94 (wherein R22a, R23a and R24a each independently represents hydrogen, C1-2alkyl or C1-2alkoxyethyl) and R21a represents a group selected from cyclopentyl, cyclohexyl, pyrrolidinyl and piperidinyl which group is linked to X5a through a carbon atom and which group may carry one substituent selected from oxo, hydroxy, halogeno, C1-2alkyl, C1-2hydroxyalkyl, C1-2alkoxy, carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl or R21a is C1-3alkyl with the proviso that when R21a is C1-3alkyl, X5a is xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94 or xe2x80x94NR23a SO2xe2x80x94); and
7) C1-2alkoxyC2-3alkyl provided that X1a is xe2x80x94Sxe2x80x94;
8) C2-3alkylX6aC2-3alkylR25a (wherein X6a represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR26aCOxe2x80x94, xe2x80x94NR27aSO2xe2x80x94, or xe2x80x94NR28axe2x80x94 (wherein R26a, R27a and R28a each independently represents hydrogen, C1-2alkyl or C1-2alkoxyethyl) and R25a represents a 5 or 6 membered saturated heterocyclic group with one or two heteroatoms, selected independently from O, S and N, which heterocyclic group may bear one or two substituents selected from oxo, hydroxy, halogeno, C1-3alkyl, C1-3hydroxyalkyl, C1-3alkoxy, carbamoyl, C1-3alkylcarbamoyl, N,N-di(C1-3alkyl)carbamoyl, C2-3alkanoyl, and C1-3alkoxycarbonyl);
9) C2-3alkylR29a (wherein R29a is piperazin-1-yl which bears at least one substituent selected from acetyl, C1-2alkoxycarbonyl, C1-2hydroxyalkyl and CONR30aR31a (wherein R30aand R31a each independently represents hydrogen or C1-2alkyl);
10) C2-3alkylR32a (wherein R32a is morpholino which may bear one or two substituents selected from oxo, C1-2alkyl, C1-2hydroxyalkyl, carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl) with the proviso that when R4a is C2-3 alkylR32a, X1a is xe2x80x94Sxe2x80x94 or xe2x80x94NR6aSO2xe2x80x94 (wherein R6a is as defined hereinbefore); and
11) C2-3alkyl (wherein 33a is morpholino which bears at least one and optionally two substituents selected from oxo, C1-2alkyl, C1-2hydroxyalkyl, carbamoyl, C1-2alkylcarbamoyl, N,N-di(C1-2alkyl)carbamoyl, acetyl and C1-2alkoxycarbonyl); and an additional value of R4a is C2-3alkylX2methylX3aR13a (wherein X2a and X3a are as define hereinbefore and R13a represents hydrogen or C1-3alkyl);]
and salts thereof.
Preferred compounds of the present invention, by virtue of their substantially equivalent activity against VEGF and EGF receptor tyrosine kinases include:
4-(4-chloro-2-fluoroanilino)-7-(1,3-dioxolan-2-ylmethoxy)-6-methoxyquinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(4-morpholinobut-2-yn-1-yloxy)quinazoline,
(E)-4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(4-morpholinobut-2-en-1-yloxy)quinazoline,
4-(4-chloro-2-fluoroanilino)-7-(3-(2,6-dimethylmorpholino)propoxy)-6-methoxyquinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(3-([N-methyl-N-methylsulphonyl]amino)propoxy)quinazoline,
7-(2-[N-tert-butoxycarbonylamino]ethoxy)-4-(4-chloro-2-fluoroanilino)-6-methoxyquinazoline,
4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(3-([N-methyl-N-methylsulphonyl]amino)propoxy)quinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-(2-oxoimidazolidin-1-yl)ethoxy)quinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-(3-oxomorpholino)ethoxy)quinazoline and
4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(2-(3-oxomorpholino)ethoxy)quinazoline
and salts thereof especially hydrochloride salts thereof.
More preferred compounds of the present invention, by virtue of their substantially equivalent activity against VEGF and EGF receptor tyrosine kinases include:
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-thiomorpholinoethoxy)quinazoline,
(S)4-(4-bromo-2-fluoroanilino)-7-(3-(2-carbamoylpyrrolidin-1-yl)propoxy)-6-methoxyquinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(3-(2-oxopyrrolidin-1-yl)propoxy)quinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-(2-oxopyrrolidin-1-yl)ethoxy)quinazoline,
(S)-7-(3-(2-carbamoylpyrrolidin-1-yl)propoxy)-4-(4-chloro-2-fluoroanilino)-6-methoxyquinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-(2-morpholinoethoxy)ethoxy)quinazoline and
4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(3-(2-oxopyrrolidin-1-yl)propoxy)quinazoline
and salts thereof especially hydrochloride salts thereof.
Particularly preferred compounds of the present invention, by virtue of their substantially equivalent activity against VEGF and EGF receptor tyrosine kinases include:
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-(2-methoxyethoxy)ethoxy)quinazoline and
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-3-yl)methoxyquinazoline and salts thereof especially hydrochloride salts thereof.
Additional particularly preferred compounds of the present invention, by virtue of their substantially equivalent activity against VEGF and EGF receptor tyrosine kinases include:
4-(4-bromo-2-fluoroanilino)-7-(3-(1,1-dioxothiomorpholino)propoxy)-6-methoxyquinazoline,
4-(bromo-2-fluoroanilino)-6-methoxy-7-(2-(2-methoxyethoxy)ethoxy)quinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-(2-pyrrolidin-1-ylethoxy)ethoxy)quinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-(2-[4-methylpiperazin-1-yl]ethoxyethoxy)quinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(3-morpholinopropylthio)quinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-(2[N-methyl-N-methoxyacetyl]amino)ethoxy)quinazoline and
4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(2-(2-oxopyrrolidin-1-yl)ethoxy)quinazoline
and salts thereof especially hydrochloride salts thereof.
Especially preferred compounds of the present invention, by virtue of their substantially equivalent activity against VEGF and EGF receptor tyrosine kinases include:
(E)-4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(4-(pyrrolidin-1-yl)but-2-en-1-yloxy)quinazoline,
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(3-(methylsulphonyl)propoxy)quinazoline,
(S)-4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-3-yl)methoxyquinazoline and
(R)-4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-3-yl)methoxyquinazoline
and salts thereof especially hydrochloride salts thereof, of which 4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(3-(methylsulphonyl)propoxy)quinazoline and salts thereof especially hydrochloride salts thereof, is more especially preferred.
In a particular aspect of the present invention preferred compounds are:
4-(3-acetoxy-4-methylanilino)-6-methoxy-7-(2-methoxyethoxy)quinazoline
4-(4-chloro-2-fluoroanilino)-7-(2-(1,3-dioxolan-2-yl)ethoxy)-6-methoxyquinazoline
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)quinazoline
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline
4-(4-chloro-2-fluoroanilino)-7-(1,3-dioxolan-2-ylmethoxy)-6-methoxyquinazoline
4-(4-chloro-2-fluoroanilino)-6-methoxy-7-(2-thiomorpholinoethoxy)quinazoline
and salts thereof especially the hydrochloride salts thereof.
For the avoidance of doubt it is to be understood that where in this specification a group is qualified by xe2x80x98hereinbefore definedxe2x80x99 or xe2x80x98defined hereinbeforexe2x80x99 the said group encompasses the first occurring and broadest definition as well as each and all of the preferred definitions for that group.
In this specification unless stated otherwise the term xe2x80x9calkylxe2x80x9d includes both straight and branched chain alkyl groups but references to individual alkyl groups such as xe2x80x9cpropylxe2x80x9d are specific for the straight chain version only. An analogous convention applies to other generic terms. Unless otherwise stated the term xe2x80x9calkylxe2x80x9d advantageously refers to chains with 1-6 carbon atoms, preferably 1-4 carbon atoms. The term xe2x80x9calkoxyxe2x80x9d as used herein unless stated otherwise includes xe2x80x9calkylxe2x80x9d xe2x80x94O-groups in which xe2x80x9calkylxe2x80x9d is as hereinbefore defined. The term xe2x80x9carylxe2x80x9d as used herein unless stated otherwise includes reference to a C6-10 aryl group which may, if desired, carry one or more substituents selected from halogeno, alkyl, alkoxy, nitro, trifluoromethyl and cyano, (wherein alkyl and alkoxy are as hereinbefore defined). The term xe2x80x9caryloxyxe2x80x9d as used herein unless otherwise stated includes xe2x80x9carylxe2x80x9d-groups in which xe2x80x9carylxe2x80x9d is as hereinbefore defined. The term xe2x80x9csulphonyloxyxe2x80x9d as used herein refers to alkylsulphonyloxy and arylsulphonyloxy groups in which xe2x80x9calkylxe2x80x9d and xe2x80x9carylxe2x80x9d are as hereinbefore defined. The term xe2x80x9calkanoylxe2x80x9d as used herein unless otherwise stated includes formyl and alkylCxe2x95x90O groups in which xe2x80x9calkylxe2x80x9d is as defined hereinbefore, for example C2alkanoyl is ethanoyl and refers to CH3Cxe2x95x90O, C1alkanoyl is formyl and refers to CHO. In this specification unless stated otherwise the term xe2x80x9calkenylxe2x80x9d includes both straight and branched chain alkenyl groups but references to individual alkenyl groups such as 2-butenyl are specific for the straight chain version only. Unless otherwise stated the term xe2x80x9calkenylxe2x80x9d advantageously refers to chains with 2-5 carbon atoms, preferably 3-5 carbon atoms. In this specification unless stated otherwise the term xe2x80x9calkynylxe2x80x9d includes both straight and branched chain alkynyl groups but references to individual alkynyl groups such as 2-butynyl are specific for the straight chain version only. Unless otherwise stated the term xe2x80x9calkynylxe2x80x9d advantageously refers to chains with 2-5 carbon atoms, preferably 3-5 carbon atoms.
In formula I, as hereinbefore defined, hydrogen will be present at positions 2 and 8 of the quinazoline group.
Within the present invention it is to be understood that a compound of the formula I or a salt thereof may exhibit the phenomenon of tautomerism and that the formulae drawings within this specification can represent only one of the possible tautomeric forms. It is to be understood that the invention encompasses any tautomeric form which inhibits VEGF receptor tyrosine kinase activity and is not to be limited merely to any one tautomeric form utilised within the formulae drawings.
It is also to be understood that certain compounds of the formula I and salts thereof can exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms which inhibit VEGF receptor tyrosine kinase activity.
For the avoidance of any doubt, it is to be understood that when X1 is, for example, a group of formula xe2x80x94NR7COxe2x80x94, it is the nitrogen atom bearing the R7 group which is attached to the quinazoline ring and the carbonyl (CO) group is attached to R4, whereas when X1 is, for example, a group of formula xe2x80x94CONR8xe2x80x94, it is the carbonyl group which is attached to the quinazoline ring and the nitrogen atom bearing the R8 group is attached to R4. A similar convention applies to the other two atom X1 linking groups such as xe2x80x94NR10SO2xe2x80x94 and xe2x80x94SO2NR9xe2x80x94. When X1 is xe2x80x94NR11xe2x80x94 it is the nitrogen atom bearing the R11 group which is linked to the quinazoline ring and to R4. An analogous convention applies to other groups, thus when R4 is, for example, a group of the formula C1-5alkylX5R27 and X5 is a group xe2x80x94NR28COxe2x80x94, it is the nitrogen atom bearing the R28 group which is attached to the alkyl chain which is attached to the quinazoline ring and the carbonyl (CO) group is attached to R27, whereas when X5 is, for example, a group of formula xe2x80x94CONR29xe2x80x94, it is the carbonyl group which is attached to the alkyl chain which is attached to the quinazoline ring and the nitrogen atom bearing the R29 group is attached to R27. It is further to be understood that when X1 represents xe2x80x94NR11xe2x80x94 and R11 is C1-3alkoxyC2-3alkyl it is the C2-3alkyl moiety which is linked to the nitrogen atom of X1 and an analogous convention applies to other groups.
For the avoidance of any doubt, it is to be understood that in a compound of the formula I when R4 is, for example, a group of formula C1-5alkylX2C1-5alkylX3R16, it is the terminal C1-5alkyl moiety which is bound to X1, similarly when R4 is, for example, a group of formula C2-5alkenylR14 it is the C2-5alkenyl moiety which is bound to X1 and an analgous convention applies to other groups. When R4 is a group 1-R33prop-1-en-3-yl it is the first carbon to which the group R33 is attached and it is the third carbon which is linked to X1, similarly when R4 is a group 2-R33pent-3-en-5-yl it is the second carbon to which the group R33 is attached and it is the fifth carbon which is linked to X1, and an analogous convention applies to other groups.
The present invention relates to the compounds of formula I as hereinbefore defined as well as to the salts thereof. Salts for use in pharmaceutical compositions will be pharmaceutically acceptable salts, but other salts may be useful in the production of the compounds of formula I and their pharmaceutically acceptable salts. Pharmaceutically acceptable salts of the invention may, for example, include acid addition salts of the compounds of formula I as hereinbefore defined which are sufficiently basic to form such salts. Such acid addition salts include for example salts with inorganic or organic acids affording pharmaceutically acceptable anions such as with hydrogen halides (especially hydrochloric or hydrobromic acid of which hydrochloric acid is particularly preferred) or with sulphuric or phosphoric acid, or with trifluoroacetic, citric or maleic acid. In addition where the compounds of formula I are sufficiently acidic, pharmaceutically acceptable salts may be formed with an inorganic or organic base which affords a pharmaceutically acceptable cation. Such salts with inorganic or organic bases include for example an alkali metal salt, such as a sodium or potassium salt, an alkaline earth metal salt such as a calcium or magnesium salt, an ammonium salt or for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
A compound of the formula I, or salt thereof, and other compounds of the invention (as hereinafter defined) may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Such processes include, for example, those illustrated in European Patent Applications, Publication Nos. 0520722, 0566226, 0602851 and 0635498. Such processes, are provided as a further feature of the invention and are as described hereinafter. Necessary starting materials may be obtained by standard procedures of organic chemistry. The preparation of such starting materials is described within the accompanying non-limiting Examples. Alternatively necessary starting materials are obtainable by analogous procedures to those illustrated which are within the ordinary skill of an organic chemist.
Thus the following processes (a) to (g) and (i) to (v) constitute further features of the present invention.
(a) Compounds of the formula I and salts thereof may be prepared by the reaction of a compound of the formula III: 
(wherein R1, R2, X1 and R4 are as defined hereinbefore and L1 is a displaceable moiety), with a compound of the formula IV: 
(wherein R3 and m are as defined hereinbefore) whereby to obtain compounds of the formula I and salts thereof. A convenient displaceable moiety L1 is, for example, a halogeno, alkoxy (preferably C1-4 alkoxy), aryloxy or sulphonyloxy group, for example a chloro, bromo, methoxy, phenoxy, methanesulphonyloxy or toluene4-sulphonyloxy group.
The reaction is advantageously effected in the presence of either an acid or a base. Such an acid is, for example, an anhydrous inorganic acid such as hydrogen chloride. Such a base is, for example, an organic amine base such as, for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, morpholine, N-methylmorpholine or diazabicyclo[5.4.0]undec-7-ene, or for example, an alkali metal or alkaline earth metal carbonate or hydroxide, for example sodium carbonate, potassium carbonate, calcium carbonate, sodium hydroxide or potassium hydroxide. Alternatively such a base is, for example, an alkali metal hydride, for example sodium hydride, or an alkali metal or alkaline earth metal amide, for example sodium amide or sodium bis(trimethylsilyl)amide. The reaction is preferably effected in the presence of an inert solvent or diluent, for example an alkanol or ester such as methanol, ethanol, 2-propanol or ethyl acetate, a halogenated solvent such as methylene chloride, trichloromethane or carbon tetrachloride, an ether such as tetrahydrofuran or 1,4-dioxan, an aromatic hydrocarbon solvent such as toluene, or a dipolar aprotic solvent such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidin-2-one or dimethylsulphoxide. The reaction is conveniently effected at a temperature in the range, for example, 10 to 150xc2x0 C., preferably in the range 20 to 80xc2x0 C.
The compound of the invention may be obtained from this process in the form of the free base or alternatively it may be obtained in the form of a salt with the acid of the formula H-L1 wherein L1 has the meaning defined hereinbefore. When it is desired to obtain the free base from the salt, the salt may be treated with a base as defined hereinbefore using a conventional procedure.
(b) Where the group of formula IIa: 
(wherein R3 and m are as hereinbefore defined) represents a phenyl group carrying one or more hydroxy groups, a compound of the formula I and salts thereof can be prepared by the deprotection of a compound of formula V: 
(wherein X1, m, R1, R2, R3 and R4 are as hereinbefore defined, P represents a phenolic hydroxy protecting group and p1 is an integer from 1 to 5 equal to the number of protected hydroxy groups and such that m-p1 is equal to the number of R3 substituents which are not protected hydroxy). The choice of phenolic hydroxy protecting group P is within the standard knowledge of an organic chemist, for example those included in standard texts such as xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d T. W. Greene and R. G. M. Wuts, 2nd Ed. Wiley 1991, including ethers (for example, methyl, methoxymethyl, allyl and benzyl and benzyl substituted with up to two substituents selected from C1-4alkoxy and nitro), silyl ethers (for example, t-butyldiphenylsilyl and t-butyldimethylsilyl), esters (for example, acetate and benzoate) and carbonates (for example, methyl and benzyl and benzyl substituted with up to two substituents selected from C1-4 alkoxy and nitro). The removal of such a phenolic hydroxy protecting group may be effected by any of the procedures known for such a transformation, including those reaction conditions indicated in standard texts such as that indicated hereinbefore, or by a related procedure. The reaction conditions preferably being such that the hydroxy derivative is produced without unwanted reactions at other sites within the starting or product compounds. For example, where the protecting group P is acetate, the transformation may conveniently be effected by treatment of the quinazoline derivative with a base as defined hereinbefore and including ammonia and its mono and di-alkylated derivatives, preferably in the presence of a protic solvent or co-solvent such as water or an alcohol, for example methanol or ethanol. Such a reaction can be effected in the presence of an additional inert solvent or diluent as defined hereinbefore and at a temperature in the range 0 to 50xc2x0 C., conveniently at about 20xc2x0 C.
(c) Production of those compounds of formula I and salts thereof wherein the substituent X1 is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94NR11xe2x80x94 or xe2x80x94SO2xe2x80x94, CONR8xe2x80x94 or xe2x80x94SO2NR9xe2x80x94 can be achieved by the reaction, conveniently in the presence of a base as defined hereinbefore, of a compound of the formula VI: 
(wherein m, X1, R1, R2 and R3 are as hereinbefore defined) with a compound of formula VII:
R4-L1xe2x80x83xe2x80x83(VII) 
(wherein R4 and L1 are as hereinbefore defined); L1 is a displaceable moiety for example a halogeno or sulphonyloxy group such as a bromo or methanesulphonyloxy group. Conveniently L1 is a group O-+P(R52)3 (wherein R52 is butyl or phenyl) and in such cases the compound of formula VII is conveniently formed in situ. The reaction is preferably effected in the presence of a base (as defined hereinbefore in process (a)) and advantageously in the presence of an inert solvent or diluent (as defined hereinbefore in process (a)), advantageously at a temperature in the range, for example 10 to 150xc2x0 C., conveniently at about 50xc2x0 C.
(d) Compounds of the formula I and salts thereof may be prepared by the reaction of a compound of the formula VIII: 
with a compound of the formula IX:
R4xe2x80x94X1xe2x80x94Hxe2x80x83xe2x80x83(IX) 
(wherein L1, R1, R2, R3, R4, m and X1 are all as hereinbefore defined). The reaction may conveniently be effected in the presence of a base (as defined hereinbefore in process (a)) and advantageously in the presence of an inert solvent or diluent (as defined hereinbefore in process (a)), advantageously at a temperature in the range, for example 10 to 150xc2x0 C., conveniently at about 100xc2x0 C.
(e) Compounds of the formula I and salts thereof wherein R4 is C1-5alkylR53, [wherein R53 is selected from one of the following three groups:
1) X7R27 (wherein X7 represents xe2x80x94Oxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94NR54COxe2x80x94, xe2x80x94NR55SO2xe2x80x94 or xe2x80x94NR56xe2x80x94 (wherein R54, R55 and R56 each independently represents hydrogen, C1-3alkyl or C1-3 alkoxy C2-3 alkyl) and R27 is as defined hereinbefore); and
2) X8C1-5 alkylX3R16 (wherein X8 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, SO2xe2x80x94, xe2x80x94NR57COxe2x80x94, xe2x80x94NR58SO2xe2x80x94 or xe2x80x94NR59xe2x80x94 (wherein R57, R58 and R59 each independently represents hydrogen, C1-3alkyl or C1-3alkoxyC2-3alkyl) and X3 and R16 are as defined hereinbefore); and
3) X9C1-5alkylR33 (wherein X9 represents xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94SO2xe2x80x94, xe2x80x94NR60COxe2x80x94, xe2x80x94NR61SO2xe2x80x94 or xe2x80x94NR62xe2x80x94 (wherein R60, R61 and R62 each independently represents hydrogen, C1-3alkyl or C1-3alkoxyl or C2-3alkyl) and R33 is as defined hereinbefore);]
may be prepared by reacting a compound of the formula X: 
(wherein L1, X1, R1, R2, R3 and m are as hereinbefore defined and R63 is C1-5alkyl) with a compound of the formula XI:
R53xe2x80x94Hxe2x80x83xe2x80x83(XI) 
(wherein R33 is as defined hereinbefore) to give a compound of the formula I. The reaction may conveniently be effected in the presence of a base (as defined hereinbefore in process (a)) and advantageously in the presence of an inert solvent or diluent (as defined hereinbefore in process (a)), and at a temperature in the range, for example 0 to 150xc2x0 C., conveniently at about 50xc2x0 C.
Compounds of the formula I wherein R4 is C2-5alkylR45, (wherein R45 is as defined hereinbefore), may be prepared by reacting a compound of formula X (wherein R63 is C2-5alkyl) with a compound of the formula XIa:
R4xe2x80x94Hxe2x80x83xe2x80x83(XIa) 
(wherein R45 is as defined hereinbefore) to give a compound of the formula I. The reaction may conveniently be effected in the presence of a base (as defined hereinbefore in process (a)) and advantageously in the presence of an inert solvent or diluent (as defined hereinbefore in process (a)), and at a temperature in the range, for example 0 to 150xc2x0 C., conveniently at about 50xc2x0 C.
(f) The production of those compounds of the formula I and salts thereof wherein the substituent R1 is represented by xe2x80x94NR5R6, where one or both of R5 and R6 are C1-3alkyl, may be effected by the reaction of compounds of formula I wherein the substituent R1 is an amino group and an alkylating agent, preferably in the presence of a base as defined hereinbefore. Such alkylating agents are C1-3alkyl moieties bearing a displaceable moiety as defined hereinbefore such as C1-3alkyl halides for example C1-3 alkyl chloride, bromide or iodide. The reaction is preferably effected in the presence of an inert solvent or diluent (as defined hereinbefore in process (a)) and at a temperature in the range, for example, 10 to 100xc2x0 C., conveniently at about ambient temperature.
(g) The production of compounds of formula I and salts thereof wherein one or more of the substituents R1, R2 or R3 is an amino group may be effected by the reduction of a corresponding compound of formula I wherein the substituent(s) at the corresponding position(s) of the quinazoline and/or aniline ring is/are a nitro group(s). The reduction may conveniently be effected as described in process (i) hereinafter. The production of a compound of formula I and salts thereof wherein the substituent(s) at the corresponding position(s) of the quinazoline and/or aniline ring is/are a nitro group(s) may be effected by the processes described hereinbefore and hereinafter in processes (a-e) and (i-v) using a quinazoline compound selected from the compounds of the formulae (I-XXVII) in which the substituent(s) at the corresponding position(s) of the quinazoline and/or aniline ring is/are a nitro group(s).
(i) The compounds of formula III and salts thereof, constitute a further feature of the present invention. Such compounds in which L1 is halogeno may for example be prepared by halogenating a compound of the formula XII: 
(wherein R1, R2, 4 and X1 are as hereinbefore defined).
Convenient halogenating agents include inorganic acid halides, for example thionyl chloride, phosphorus(III)chloride, phosphorus(V)oxychloride and phosphorus(V)chloride. The halogenation reaction is conveniently effected in the presence of an inert solvent or diluent such as for example a halogenated solvent such as methylene chloride, trichloromethane or carbon tetrachloride, or an aromatic hydrocarbon solvent such as benzene or toluene. The reaction is conveniently effected at a temperature in the range, for example 10 to 150xc2x0 C., preferably in the range 40 to 100xc2x0 C.
The compounds of formula XII and salts thereof which constitute a further feature of the present invention may for example be prepared by reacting a compound of the formula XIII: 
(wherein R1, R2 and L1 are as hereinbefore defined) with a compound of the formula IX as hereinbefore defined. The reaction may conveniently be effected in the presence of a base (as defined hereinbefore in process (a)) and advantageously in the presence of an inert solvent or diluent (as defined hereinbefore in process (a)), advantageously at a temperature in the range, for example 10 to 150xc2x0 C., conveniently at about 100xc2x0 C.
The compounds of formula XII and salts thereof may also be prepared by cyclising a compound of the formula XIV: 
(wherein R1, R2, R4 and X1, are as hereinbefore defined, and A1 is an hydroxy, alkoxy (preferably C1-4alkoxy) or amino group) whereby to form a compound of formula XII or salt thereof. The cyclisation may be effected by reacting a compound of the formula XIV, where A1 is an hydroxy or alkoxy group, with formamide or an equivalent thereof effective to cause cyclisation whereby a compound of formula XII or salt thereof is obtained, such as [3-(dimethylamino)-2-azaprop-2-enylidene]dimethylammonium chloride. The cyclisation is conveniently effected in the presence of formnamide as solvent or in the presence of an inert solvent or diluent such as an ether for example 1,4-dioxan. The cyclisation is conveniently effected at an elevated temperature, preferably in the range 80 to 200xc2x0 C. The compounds of formula XII may also be prepared by cyclising a compound of the formula XIV, where A1 is an amino group, with formic acid or an equivalent thereof effective to cause cyclisation whereby a compound of formula XII or salt thereof is obtained. Equivalents of formic acid effective to cause cyclisation include for example a tri-C1-4alkoxymethane, for example triethoxymethane and trimethoxymethane. The cyclisation is conveniently effected in the presence of a catalytic amount of an anhydrous acid, such as a sulphonic acid for example p-toluenesulphonic acid, and in the presence of an inert solvent or diluent such as for example a halogenated solvent such as methylene chloride, trichloromethane or carbon tetrachloride, an ether such as diethylether or tetrahydrofuran, or an aromatic hydrocarbon solvent such as toluene. The cyclisation is conveniently effected at a temperature in the range, for example 10 to 100xc2x0 C., preferably in the range 20 to 50xc2x0 C.
Compounds of formula XIV and salts thereof, which constitute a further feature of the present invention, may for example be prepared by the reduction of the nitro group in a compound of the formula XV: 
(wherein R1, R2, R4, X1 and A1 are as hereinbefore defined) to yield a compound of formula XIV as hereinbefore defined. The reduction of the nitro group may conveniently be effected by any of the procedures known for such a transformation. The reduction may be carried out, for example, by the hydrogenation of a solution of the nitro compound in the presence of an inert solvent or diluent as defined hereinbefore in the presence of a metal effective to catalyse hydrogenation reactions such as palladium or platinum. A further reducing agent is, for example, an activated metal such as activated iron (produced for example by washing iron powder with a dilute solution of an acid such as hydrochloric acid). Thus, for example, the reduction may be effected by heating the nitro compound and the activated metal in the presence of a solvent or diluent such as a mixture of water and alcohol, for example methanol or ethanol, to a temperature in the range, for example 50 to 150xc2x0 C., conveniently at about 70xc2x0 C.
Compounds of the formula XV and salts thereof which constitute a further feature of the present invention, may for example be prepared by the reaction of a compound of the formula XVI: 
(wherein R1, R2, L1 and A1 are as hereinbefore defined) with a compound of the formula IX as hereinbefore defined to give a compound of the formual XV. The reaction of the compounds of formulae XVI and IX is conveniently effected under conditions as described for process (d) hereinbefore.
Compounds of formula XV and salts thereof, may for example also be prepared by the reaction of a compound of the formula XVII: 
(wherein R1, R2, X1 and A1 are as hereinbefore defined with the proviso that X1 is not xe2x80x94CH2xe2x80x94) with a compound of the formula VII as hereinbefore defined to yield a compound of formula XV as hereinbefore defined. The reaction of the compounds of formulae XVII and VII is conveniently effected under conditions as described for process (c) hereinbefore.
The compounds of formula III and salts thereof may also be prepared for example by reacting a compound of the formula XVIII: 
(wherein R1, R2 and X1 are as hereinbefore defined with the proviso that X1 is not xe2x80x94CH2xe2x80x94 and L2 represents a displaceable protecting moiety) with a compound of the formula VII as hereinbefore defined, whereby to obtain a compound of formula III in which L1 is represented by L2.
A compound of formula XVIII is conveniently used in which L2 represents a phenoxy group which may if desired carry up to 5 substituents, preferably up to 2 substituents, selected from halogeno, nitro and cyano. The reaction may be conveniently effected under conditions as described for process (c) hereinbefore.
The compounds of formula XVIII and salts thereof as hereinbefore defined may for example be prepared by deprotecting a compound of the formula XIX: 
(wherein R1, R2, P, X1 and L2 are as hereinbefore defined with the proviso that X1 is not xe2x80x94CH2xe2x80x94). Deprotection may be effected by techniques well known in the literature, for example where P represents a benzyl group deprotection may be effected by hydrogenolysis or by treatment with trifluoroacetic acid.
One compound of formula III may if desired be converted into another compound of formula III in which the moiety L1 is different. Thus for example a compound of formula III in which L1 is other than halogeno, for example optionally substituted phenoxy, may be converted to a compound of formula III in which L1 is halogeno by hydrolysis of a compound of formula III (in which L1 is other than halogeno) to yield a compound of formula XII as hereinbefore defined, followed by introduction of halide to the compound of formula XII, thus obtained as hereinbefore defined, to yield a compound of formula III in which L1 represents halogen.
(ii) The compounds of formula V and salts thereof, constitute a further feature of the present invention, and may for example be prepared by the reaction of a compound of formula III as hereinbefore defined with a compound of the formula XX: 
(wherein R3, m, p1 and P are as hereinbefore defined). The reaction may for example be effected as described for process (a) hereinbefore.
The compounds of formula V and salts thereof may also be prepared by reacting a compound of formula XXI: 
(wherein R1, R2, L1, R3, m, p1 and P are as hereinbefore defined) with a compound of formula IX as hereinbefore defined. The reaction may for example be effected as described for process (d) above.
The compounds of formula V and salts thereof may also be prepared by reacting a compound of formula XXII: 
(wherein R1, R2, R3, X1, P, p1 and m are as hereinbefore defined with the proviso that X1 is not xe2x80x94CH2xe2x80x94) with a compound of the formula VII as hereinbefore defined. The reaction may for example be effected as described for process (c) hereinbefore.
The compounds of formula XXI and salts thereof may for example be prepared by reaction of a compound of formula XXIII: 
(wherein R1, R2, and L1 are as hereinbefore defined, and LI in the 4- and 7-positions may be the same or different) with a compound of the formula XX as hereinbefore defined. The reaction may be effected for example by a process as described in (a) above.
Compounds of the formula XXII and salts thereof may be made by reacting compounds of the formulae XIX and XX as hereinbefore defined, under conditions described in (a) hereinbefore, to give a compound of formula XXIV: 
(wherein R1, R2, R3, P, X1, p1 and m are as hereinbefore defined with the proviso that X1 is not xe2x80x94CH2xe2x80x94) and then deprotecting the compound of formula XXIV for example as described in (i) above.
(iii) Compounds of the formula VI as hereinbefore defined and salts thereof may be made by deprotecting the compound of formula XXV: 
(wherein R1, R2, R3, P, X1 and m are as hereinbefore defined) by a process for example as described in (i) above.
Compounds of the formula XXV and salts thereof may be made by reacting compounds of the formulae XIX and IV as hereinbefore defined, under the conditions described in (a) hereinbefore, to give a compound of the formula XXV or salt thereof.
(iv) Compounds of the formula VIII and salts thereof as hereinbefore defined may be made by reacting compounds of the formulae XXIII and IV as hereinbefore defined, the reaction for example being effected by a process as described in (a) above.
(v) Compounds of the formula X as defined hereinbefore and salts thereof may for example be made by the reaction of a compound of formula VI as defined hereinbefore with a compound of the formula XXVI:
L1xe2x80x94R63xe2x80x94L1xe2x80x83xe2x80x83(XXVI) 
(wherein L1 and R63 are as hereinbefore defined) to give a compound of the formula X. The reaction may be effected for example by a process as described in (c) above.
Compounds of the formula X and salts thereof may also be made for example by deprotecting a compound of the formula XXVII: 
(wherein L1, R63, X1, R1, R2, R3, P, m and p1 are as defined hereinbefore) by a process for example as described in (b) above.
Compounds of the formula XXVII and salts thereof may be made for example by reacting compounds of the formulae XXII and XXVI as defined hereinbefore, under the conditions described in (c) above.
When a pharmaceutically acceptable salt of a compound of the formula I is required, it may be obtained, for example, by reaction of said compound with, for example, an acid using a conventional procedure, the acid having a pharmaceutically acceptable anion.
Many of the intermediates defined herein are novel, for example, those of the formulae III, V, XII, XIV and XV, and these are provided as a further feature of the invention.
Intermediates of the formulae VIII, X, XXI, XXII, XXIV, XXV and XXVII are also provided as a further feature of the invention.
The identification of compounds which potently inhibit the tyrosine kinase activity associated with the VEGF receptors such as Flt and/or KDR and which inhibit angiogenesis and/or increased vascular permeability is desirable and is the subject of the present invention. These properties may be assessed, for example, using one or more of the procedures set out below:
(a) In Vitro Receptor Tyrosine Kinase Inhibition Test
This assay determines the ability of a test compound to inhibit tyrosine kinase activity. DNA encoding VEGF or epidermal growth factor (EGF) receptor cytoplasmic domains may be obtained by total gene synthesis (Edwards M, International Biotechnology Lab 5(3), 19-25, 1987) or by cloning. These may then be expressed in a suitable expression system to obtain polypeptide with tyrosine kinase activity. For example VEGF and EGF receptor cytoplasmic domains, which were obtained by expression of recombinant protein in insect cells, were found to display intrinsic tyrosine kinase activity. In the case of the VEGF receptor Flt (Genbank accession number X51602), a 1.7 kb DNA fragment encoding most of the cytoplasmic domain, commencing with methionine 783 and including the termination codon, described by Shibuya et al (Oncogene, 1990, 5: 519-524), was isolated from cDNA and cloned into a baculovirus transplacement vector (for example pAcYMI (see The Baculovirus Expression System: A Laboratory Guide, L. A. King and R. D. Possee, Chapman and Hall, 1992) or pAc360 or pBlueBacHis (available from Invitrogen Corporation)). This recombinant construct was co-transfected into insect cells (for example Spodoptera frugiperda 21(Sf21)) with viral DNA (eg Pharmingen BaculoGold) to prepare recombinant baculovirus. (Details of the methods for the assembly of recombinant DNA molecules and the preparation and use of recombinant baculovirus can be found in standard texts for example Sambrook et al, 1989, Molecular cloningxe2x80x94A Laboratory Manual, 2nd edition, Cold Spring Harbour Laboratory Press and O""Reilly et al, 1992, Baculovirus Expression Vectorsxe2x80x94A Laboratory Manual, W. H. Freeman and Co, New York). For other tyrosine kinases for use in assays, cytoplasmic fragments starting from methionine 806 (KDR, Genbank accession number L04947) and methionine 668 (EGF receptor, Genbank accession number X00588) may be cloned and expressed in a similar manner.
For expression of cFlt tyrosine kinase activity, Sf12 cells were infected with plaque-pure cFlt recombinant virus at a multiplicity of infection of 3 and harvested 48 hours later. Harvested cells were washed with ice cold phosphate buffered saline solution (PBS) (10 mM sodium phosphate pH7.4, 138 mM sodium chloride, 2.7 mM potassium chloride) then resuspended in ice cold HNTG/PMSF (20 mM Hepes pH7.5, 150 mM sodium chloride, 10% v/v glycerol, 1% v/v Triton X100, 1.5 mM magnesium chloride, 1 mM ethylene glycol-bis(xcex2aminoethyl ether) N, N, Nxe2x80x2,Nxe2x80x2-tetraacetic acid (EGTA), 1 mM PMSF (phenylmethylsulphonyl fluoride); the PMSF is added just before use from a freshly-prepared 100 mM solution in methanol) using 1 ml HNTG/PMSF per 10 million cells. The suspension was centrifuged for 10 minutes at 13,000 rpm at 4xc2x0 C., the supernatant (enzyme stock) was removed and stored in aliquots at xe2x88x9270xc2x0 C. Each new batch of stock enzyme was titrated in the assay by dilution with enzyme diluent (100 mM Hepes pH 7.4, 0.2 mM sodium orthovanadate, 0.1% v/v Triton X100, 0.2 mM dithiothreitol). For a typical batch, stock enzyme is diluted 1 in 2000 with enzyme diluent and 50 xcexcl of dilute enzyme is used for each assay well.
A stock of substrate solution was prepared from a random copolymer containing tyrosine, for example Poly (Glu, Ala, Tyr) 6:3:1 (Sigma P3899), stored as 1 mg/ml stock in PBS at xe2x88x9220xc2x0 C. and diluted 1 in 500 with PBS for plate coating.
On the day before the assay 100 xcexcl of diluted substrate solution was dispensed into all wells of assay plates (Nunc maxisorp 96-well immunoplates) which were sealed and left overnight at 4xc2x0 C.
On the day of the assay the substrate solution was discarded and the assay plate wells were washed once with PBST (PBS containing 0.05% v/v Tween 20) and once with 5 mM Hepes pH7.4.
Test compounds were diluted with 10% dimethylsulphoxide (DMSO) and 25 xcexcl of diluted compound was transferred to wells in the washed assay plates. xe2x80x9cTotalxe2x80x9d control wells contained 10% DMSO instead of compound. Twenty five microliters of 40 mM manganese(II)chloride containing 8 xcexcM adenosine-5-triphosphate (ATP) was added to all test wells except xe2x80x9cblankxe2x80x9d control wells which contained manganese(II)chloride without ATP. To start the reactions 50 xcexcl of freshly diluted enzyme was added to each well and the plates were incubated at room temperature for 20 minutes. The liquid was then discarded and the wells were washed twice with PBST. One hundred microliters of mouse IgG anti-phosphotyrosine antibody (Upstate Biotechnology Inc. product 05-321), diluted 1 in 6000 with PBST containing 0.5% w/v bovine serum albumin (BSA), was added to each well and the plates were incubated for 1 hour at room temperature before discarding the liquid and washing the wells twice with PBST. One hundred microliters of horse radish peroxidase (HRP)linked sheep anti-mouse Ig antibody (Amersham product NXA 931), diluted 1 in 500 with PBST containing 0.5% w/A BSA, was added and the plates were incubated for 1 hour at room temperature before discarding the liquid and washing the wells twice with PBST. One hundred microliters of 2,2xe2x80x2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) solution, freshly prepared using one 50 mg ABTS tablet (Boehringer 1204 521) in 50 ml freshly prepared 50 mM phosphate-citrate buffer pH5.0+0.03% sodium perborate (made with 1 phosphate citrate buffer with sodium perborate (PCSB) capsule (Sigma P4922) per 100 ml distilled water), was added to each well. Plates were then incubated for 20-60 minutes at room temperature until the optical density value of the xe2x80x9ctotalxe2x80x9d control wells, measured at 405 nm using a plate reading spectrophotometer, was approximately 1.0. xe2x80x9cBlankxe2x80x9d (no ATP) and xe2x80x9ctotalxe2x80x9d (no compound) control values were used to determine the dilution range of test compound which gave 50% inhibtion of enzyme activity.
(b) In Vitro HUVEC Proliferation Assay
This assay determines the ability of a test compound to inhibit the growth factor-stimulated proliferation of human umbilical vein endothelial cells (HUVEC).
HUVEC cells were isolated in MCDB 131 (Gibco BRL)+7.5% v/v foetal calf serum (FCS) and were plated out (at passage 2 to 8), in MCDB 131+2% v/v FCS+3 xcexcg/ml heparin+1 xcexcg/ml hydrocortisone, at a concentration of 1000 cells/well in 96 well plates. After a minimum of 4 hours they were dosed with the appropriate growth factor (i.e. VEGF 3ng/ml, EGF 3 ng/ml or b-FGF 0.3 ng/ml) and compound. The cultures were then incubated for 4 days at 37xc2x0 C. with 7.5% carbon dioxide. On day 4 the cultures were pulsed with 1 xcexcCi/well of tritiated-thymidine (Amersham product TRA 61) and incubated for 4 hours. The cells were harvested using a 96-well plate harvester (Tomtek) and then assayed for incorporation of tritium with a Beta plate counter. Incorporation of radioactivity into cells, expressed as cpm, was used to measure inhibition of growth factor-stimulated cell proliferation by compounds.
(c)In Vivo Rat Uterine Oedema Assay
This test measures the capacity of compounds to reduce the acute increase in uterine weight in rats which occurs in the first 4-6 hours following oestrogen stimulation. This early increase in uterine weight has long been known to be due to oedema caused by increased permeability-of the uterine vasculature and recently Cullinan-Bove and Koos (Endocrinology, 1993,133:829-837) demonstrated a close temporal relationship with increased expression of VEGF mRNA in the uterus. We have found that prior treatment of the rats with a neutralising monoclonal antibody to VEGF significantly reduces the acute increase in uterine weight, confirming that the increase in weight is substantially mediated by VEGF.
Groups of 20 to 22-day old rats were treated with a single subcutaneous dose of oestradiol benzoate (2.5 xcexcg/rat) in a solvent, or solvent only. The latter served as unstimulated controls. Test compounds were orally administered at various times prior to the administration of oestradiol benzoate. Five hours after the administration of oestradiol benzoate the rats were humanely sacrificed and their uteri were dissected, blotted and weighed. The increase in uterine weight in groups treated with test compound and oestradiol benzoate and with oestradiol benzoate alone was compared using a Student T test. Inhibition of the effect of oestradiol benzoate was considered significant when p less than 0.05.
According to a further aspect of the invention there is provided a pharmaceutical composition which comprises a compound of the formula I as defined hereinbefore or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable excipient or carrier.
The composition may be in a form suitable for oral administration, for example as a tablet or capsule, for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) for example as a sterile solution, suspension or emulsion, for topical administration for example as an ointment or cream or for rectal administration for example as a suppository. In general the above compositions may be prepared in a conventional manner using conventional excipients.
The compositions of the present invention are advantageously presented in unit dosage form. The compound will normally be administered to a warm-blooded animal at a unit dose within the range 5-5000 mg per square meter body area of the animal, i.e. approximately 0.1-100 mg/kg. A unit dose in the range, for example, 1-100 mg/kg. preferably 1-10 mg/kg is envisaged and this normally provides a therapeutically-effective dose. A unit dose form such as a tablet or capsule will usually contain, for example 1-250 mg of active ingredient.
According to a further aspect of the present invention there is provided a compound of the formula I or a pharmaceutically acceptable salt thereof as defined hereinbefore for use in a method of treatment of the human or animal body by therapy.
We have found that compounds of the present invention inhibit VEGF receptor tyrosine kinase activity and are therefore of interest for their antiangiogenic effects and/or their ability to cause a reduction in vascular permeability.
A further feature of the present invention is a compound of formula I, or a pharmaceutically acceptable salt thereof, for use as a medicament, conveniently a compound of formula I, or a pharmaceutically acceptable salt thereof, for use as a medicament for producing an antiangiogenic and/or vascular permeability reducing effect in a warm-blooded animal such as a human being.
Thus according to a further aspect of the invention there is provided the use of a compound of the formula I, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the production of an antiangiogenic and/or vascular permeability reducing effect in a warm-blooded animal such as a human being.
According to a further feature of the invention there is provided a method for producing an antiangiogenic and/or vascular permeability reducing effect in a warm-blooded animal, such as a human being, in need of such treatment which comprises administering to said animal an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof as defined hereinbefore.
As stated above the size of the dose required for the therapeutic or prophylactic treatment of a particular disease state will necessarily be varied depending on the host treated, the route of administration and the severity of the illness being treated. Preferably a daily dose in the range of 1-50 mg/kg is employed. However the daily dose will necessarily be varied depending upon the host treated, the particular route of administration, and the severity of the illness being treated. Accordingly the optimum dosage may be determined by the practitioner who is treating any particular patient.
The antiangiogenic and/or vascular permeability reducing treatment defined hereinbefore may be applied as a sole therapy or may involve, in addition to a compound of the invention, one or more other substances and/or treatments. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment. In the field of medical oncology it is normal practice to use a combination of different forms of treatment to treat each patient with cancer. In medical oncology the other component(s) of such conjoint treatment in addition to the antiangiogenic and/or vascular permeability reducing treatment defined hereinbefore may be: surgery, radiotherapy or chemotherapy. Such chemotherapy may cover three main categories of therapeutic agent:
(i) other antiangiogenic agents that work by different mechanisms from those defined hereinbefore (for example linomide, inhibitors of integrin xcex1vxcex23 function, angiostatin, razoxin, thalidomide);
(ii) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene, iodoxyfene), progestogens (for example megestrol acetate), aromatase inhibitors (for example anastrozole, letrazole, vorazole, exemestane), antiprogestogens, antiandrogens (for example flutamide, nilutamide, bicalutamide, cyproterone acetate), LHRH agonists and antagonists (for example goserelin acetate, luprolide), inhibitors of testosterone 5xcex1-dihydroreductase (for example finasteride), anti-invasion agents (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function) and inhibitors of growth factor function, (such growth factors include for example platelet derived growth factor and hepatocyte growth factor such inhibitors include growth factor antibodies, growth factor receptor antibodies, tyrosine kinase inhibitors and serine/threonine kinase inhibitors); and
(iii) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as antimetabolites (for example antifolates like methotrexate, fluoropyrimidines like 5-fluorouracil, purine and adenosine analogues, cytosine arabinoside); antitumour antibiotics (for example anthracyclines like doxorubicin, daunomycin, epirubicin and idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum derivatives (for example cisplatin, carboplatin); alkylating agents (for example nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide, nitrosoureas, thiotepa); antimitotic agents (for example vinca alkaloids like vincristine and taxoids like taxol, taxotere); topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan).
As stated above the compounds defined in the present invention are of interest for their antiangiogenic and/or vascular permeability reducing effects. Such compounds of the invention are expected to be useful in a wide range of disease states including cancer, diabetes, psoriasis, rheumatoid arthritis, Kaposi""s sarcoma, haemangioma, acute and chronic nephropathies, atheroma, arterial restenosis, autoimmune diseases, acute inflammation and ocular diseases with retinal vessel proliferation. In particular such compounds of the invention are expected to slow advantageously the growth of primary and recurrent solid tumours of, for example, the colon, breast, prostate, lungs and skin. More particularly such compounds of the invention are expected to inhibit the growth of those primary and recurrent solid tumours which are associated with VEGF especially those tumours which are significantly dependent on VEGF for their growth and spread, including for example, certain tumours of the colon, breast, prostate, lung, vulva and skin.
In addition to their use in therapeutic medicine the compounds of formula I and their pharmaceutically acceptable salts are also useful as pharmacological tools in the development and standardisation of in vitro and in vivo test systems for the evaluation of the effects of inhibitors of VEGF receptor tyrosine kinase activity in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.
It is to be understood that where the term xe2x80x9cetherxe2x80x9d is used anywhere in this specification it refers to diethyl ether.
The invention will now be illustrated in the following non-limiting Examples in which, unless otherwise stated:
(i) evaporations were carried out by rotary evaporation in vacuo and work-up procedures were carried out after removal of residual solids such as drying agents by filtration;
(ii) operations were carried out at ambient temperature, that is in the range 18-25xc2x0 C. and under an atmosphere of an inert gas such as argon;
(iii) column chromatography (by the flash procedure) and medium pressure liquid chromatography (MPLC) were performed on Merck Kieselgel silica (Art. 9385) or Merck Lichroprep RP-18 (Art. 9303) reversed-phase silica obtained from E. Merck, Darmstadt, Germany;
(iv) yields are given for illustration only and are not necessarily the maximum attainable;
(v) melting points are uncorrected and were determined using a Mettler SP62 automatic melting point apparatus, an oil-bath apparatus or a Koffler hot plate apparatus.
(vi) the structures of the end-products of the formula I were confirmed by nuclear (generally proton) magnetic resonance (NMR) and mass spectral techniques; proton magnetic resonance chemical shift values were measured on the delta scale and peak multiplicities are shown as follows: s, singlet; d, doublet; t, triplet; m, muhiplet; br, broad; q, quartet;
(vii) intermediates were not generally fully characterised and purity was assessed by thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), infra-red (IR) or NMR analysis;
(viii) the following abbreviations have been used:
DMF N, N-dimethylformamide
DMSO dimethylsulphoxide
THF tetrahydrofuran
TFA trifluoroacetic acid
NMP 1-methyl-2-pyrrolidinone.]